We report the cloning of cDNAs for rat liver apolipoprotein B (apo B) and the use of the cloned sequences to examine apo B expression at the level of mRNA in rat tissues. Fifteen putative apo B clones were identified by antibody screening of a rat liver cDNA library in the Xgtll expression vector. The identity of the clones was confirmed by immunological studies of the fusion protein products. All clones appear to contain sequences found only in apo B PI, the high molecular weight form of rat liver apo B. Blotting studies show that the clones hybridize to a single 20-kilobase liver mRNA species, sufficiently large to encode the entire apo B PI peptide, which is estimated to be 400 kDa in size. Apo B PI mRNA is abundant in liver and present in lower amounts in intestine but is absent in a variety of other tissues examined. This tissue distribution is consistent with that expected from studies on the in vivo synthesis of apo B. One clone, corresponding to a 240-base segment of the apo B PI mRNA, was sequenced and found to exhibit homology with a short region of rat apo E mRNA. Analysis of the secondary structure of the corresponding peptide did not show the preponderance of amphipathic a-helical structures characteristic of other apolipoproteins examined thus far.Apolipoprotein B (apo B) is a major protein component of mammalian very low density lipoproteins (VLDL) and chylomicrons and essentially the sole protein in low density lipoproteins (LDL). It is essential for the assembly/secretion of chylomicrons and VLDL, since these lipoproteins are absent in individuals with abetalipoproteinemia, a genetic disorder in which apo B is not produced (1). Apo B also functions as the ligand for the removal of LDL from the circulation by receptor-mediated uptake into a variety ofcells (2, 3).Despite intensive study, the structure of apo B has remained an enigma. It is among the largest peptides known, with estimates ranging from 250 to 550 kDa (1, 4-10), and is extremely hydrophobic as well as very sensitive to proteolytic degradation, and only recently has limited amino acid sequence information become available (11). Moreover, at least two major forms ofapo B are present in the circulation (8,9,(12)(13)(14)(15). In humans and most other mammals, the liver incorporates a 400-kDa species into VLDL, whereas the intestine incorporates a 210-kDa species into chylomicrons. However, the rat and mouse are exceptional in that their livers produce VLDL with comparable amounts ofboth these peptides (9,(13)(14)(15). We call these high and low molecular weight forms of apo B P(eptide)I and P(eptide)III, respectively; they are the B-100 and B-48 species in Kane's nomenclature (1,8,9). Although in the rat, PI is also accompanied by a slightly smaller component, PII, the two are very similar (9) and will be collectively referred to as PI. Peptide fingerprinting and immunological analysis suggest that PI encompasses a unique, PI-specific moiety joined to a second moiety which is identical or very similar to PIII (9,(16)(17)(18). Th...

In an effort to elucidate the mechanisms of conjugal plasmid transfer in Streptococcus faecalis, a genetic analysis of the sex pheromone-dependent tetracycline resistance plasmid pCF-10 was initiated. Rare transconjugants obtained from short matings with wild-type donors not exposed to sex pheromones were screened for increased donor potential in a subsequent mating. From this screening, a mutant plasmid, designated pCF-11, whose transfer functions are expressed in the absence of pheromone induction was isolated. Cells carrying pCF-11 spontaneously clump when grown in broth culture but do not excrete sex pheromones active against wild-type donors. In the course of initial experiments, it was observed that physiological conditions could affect plasmid transfer frequency. Therefore, a set of standardized optimal mating conditions was defined. The experiments carried out to determine these conditions revealed that a transient increase in transfer frequency of about 2 orders of magnitude occurred in early-exponential-phase donor cells. This peak of activity is independent of sex pheromone response, since it was observed with induced or uninduced donor cells carrying either pCF-11 or pCF-10.In Streptococcusfaecalis, donor cell aggregation and transfer functions of certain conjugative plasmids are induced by sex pheromones, or clumping-inducing agents (CIA), produced by recipient cells (2)(3)(4). Most of the experimental work on sex pheromone-dependent plasmid transfer has been carried out with hemolysin and bacteriocin plasmids (24). We recently reported the identification of a CIA-dependent R factor carrying tetracycline resistance (Tetr) (5).This plasmid, designated pCF-10, is a useful model for a genetic study of conjugation since it carries a selectable marker on a plasmid whose transfer genes can be turned on and off by the addition or removal of CIA. We have begun to isolate a series of mutant derivatives of this plasmid and to analyze the effects of the mutations on the donor phenotype. In the course of these initial genetic studies, occasional variations in plasmid transfer efficiencies which appeared to be related to physiological conditions of the cells in the mating mixtures were observed. In light of these observations, we felt that it would be important to define optimal mating conditions for the demonstration of differences in mating behavior between wild-type and mutant donors. In this paper, we describe the isolation of a mutant plasmid which exhibits a high donor potential in the absence of CIA. We t Present address: Department of Microbiology, University of Michigan, Ann Arbor, MI 48109.also discuss experiments in which we employed the wild-type and mutant plasmids to determine the effects of growth conditions and cell density on plasmid transfer and CIA response. MATERIALS AND METHODSBacteria and growth medium. The strains used in this study are listed in Table 1. BYGT medium (5) was employed in all experiments.Mating experiments. For the initial experiments described in this paper, donor and recipi...